The secondary structure characteristics of the hen oviduct mRNA for ovalbumin and ovomucoid will be compared for mRNA in solution, in the preinitiation complex for protein synthesis, and in the cell. Specific mRNA fragments will be produced by RNase H digestion in the presence of specific oligodeoxynucleotides. The secondary structure of each mRNA fragment will be determined by a combination of nucleotide sequence analysis of S1 nuclease resistant oligonucleotides (compared with unfragmented mRNA) and partial digestion of (32P) end labelled mRNA fragments with S1 and T1 nucleases. mRNAs crosslinked in solution with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) will be similarly analyzed for secondary structure. The ability of crosslinked vs. normal mRNAs to form preinitiation complexes with the wheat germ 40S ribosomal subunit will be measured at different potassium ion concentrations to determine whether structural changes in the mRNA molecule are necessary for recognition of the specific initiation site. In addition, mRNAs bound in a preinitiation complex will be crosslinked with AMT, extracted, and their secondary structure analyzed for changes produced by possible base pairing with 18S rRNA in the preinitiation complex. mRNA crosslinked with AMT in situ using fresh hen oviduct tissue and in isolated polysomes will be extracted and analyzed for secondary structure for comparison with mRNA secondary structure in solution and in preinitiation complexes formed in vitro. These experiments will provide a molecular basis for understanding eucaryotic mRNA structure and its effect on mRNA function as a template for protein synthesis. In a population of cellular mRNAs, the amount of protein synthesized from a specific mRNA species reflects both the amount of that mRNA synthesized and processed in the nucleus and its stability and initiation effeciency relative to other mRNAs in the cytoplasm. These cytoplasmic parameters may be at least in part a function of mRNA structure which may therefore contribute to the regulation of gene expression at the level of protein synthesis.